The proposed research is designed to characterize key genetic changes that are most likely to precipitate compensatory evolution. The approach is to investigate the effect on evolution altering a diversity of well characterized proteins in Bacillus subtilis (including ribosomal proteins and subunits of RNA polymerase and DNA gyrase). The strategy for assaying evolutionary effects is based on the following premise: in order to bring about evolutionary change, a genetic change must alter the relative fitnesses of allelic variants at other loci. One assay of each mutation's effect on evolution will be the extent to which it alters the relative fitnesses of B. subtilis strains collected from nature. Another assay will be the extent to which each mutation causes selection to favor subsequent mutations that would ordinarily have been deleterious or neutral. Comparisons of the effects on evolution of different protein alterations will provide tests of the following hypotheses: A genetic change is most likely to provoke further evolutionary responses if it 1) effects a loss in some component of fitness, 2) involves a protein that is in direct contact with many other proteins, or 3) involves a protein required in the early stages of a self-assembly process. This study will also investigate the likelihood of major mutations contributing to selection responses. The approch is to determine whether a phenotypic change brought about by a single genetic change has a greater deleterious effect than the same amount of phenotypic change conferred by many genes of small effect. Since the protein alterations of this study all confer antibiotic resistance, this work may ultimately help to determine the effect of acquiring antibiotic resistance of the future evolution of pathogenic microbes.